An affinity chromatographic approach was used to investigate the plasmin activity observed in bovine acid whey. Plasminogen-removal gel with tranexamic acid as a ligand was used for selective binding of plasminogen and plasmin through their kringle domains. Subsequently, ε-aminocaproic acid was used for elution. Plasmin activity was separated into a bound and non-bound fraction. Surprisingly, only 25% of the total activity in the acid whey was bound to the column, while 50% of the total activity was in the non-bound fraction, and a further 25% of the total activity was unaccounted for. Proteins in the bound fraction were analysed using SDS-PAGE and MALDI-MS/MS fingerprinting analysis, with plasminogen identified. Plasmin activity was purified by a factor of 4313 from the acid whey. The non-bound fraction was subjected to cation-exchange chromatography and zymogram gel analysis and showed the presence of plasminogen-derived proteolytic products such as midi-plasmin, mini-plasmin, and micro-plasmin.